In the food industry, dairy industry, and other industries, methods for rapidly detecting microorganisms with high sensitivity are very important, for example, in the control of environmental microorganisms such as the detection of microorganisms at food manufacturing plants for prevention of food poisoning, and in the inspection for contamination of microorganisms in food products (e.g., dairy products such as milk). In conventional methods for detecting living cells using a nutrient medium, it takes several days until a living microorganism is counted.
For the detection of microorganisms as described above, a method of utilizing ATP present in all organisms has been examined. As a method for detecting ATP, a bioluminescence assay employing firefly luciferase has been known. This method is an established technique for measuring ATP (see DeLuca, M. and W. D. McElroy, “Kinetics of the firefly luciferase catalyzed reactions,” Biochemistry, vol. 26. pp. 921-925 (1974)) and has been used as a rapid, hygiene monitoring (Bautista, D. A. et al., “Adenosine triphosphate bioluminescence as a method to determine microbial levels in scald and chill tanks at a poultry abattoir,” Poult. Sci., vol. 73, pp. 1673-1678 (1994)). Furthermore, an ATP assay has recently been proposed as a technology for countering bioterrorism (Spencer, R. C. and N. F. Lightfoot, “Preparedness and response to bioterrorism,” J. Infect., vol. 43, pp. 104-110 (2001)).
However, the conventional methods for assaying ATP have a detection limit (e.g., approximately 104 E. coli colony-forming units (CFU)/assay). Such a sensitivity is not sensitive enough for industrial or practical applications.
A computer simulation has suggested that ATP amplification employing adenylate kinase (ADK) and pyruvate kinase (PVK) provides a possibility that a very low level of ATP can be detected without using a photometer having high sensitivity (Chittock, R. S. et al., “Kinetic aspects of ATP amplification reactions,” Anal. Biochem, vol. 255, pp. 120-126 (1998)). However, this method has not been utilized in practice.
In order to assay a trace amount of ATP, a method for amplifying ATP has been proposed (Japanese Laid-Open Patent Publication No. 2001-299390). This method disclosed in Japanese Laid-Open Patent Publication No. 2001-299390 will be described with reference to FIG. 1. In FIG. 1, ADK refers to adenylate kinase, polyP refers to polyphosphate, and PPK refers to polyphosphate kinase. Hereinafter, these abbreviations sometimes are also used in the present specification. FIG. 1a shows that in the absence of ATP, ATP is not theoretically produced from AMP and polyphosphate. As shown in FIG. 1b, in the presence of ATP, ADK causes a transphosphorylation from ATP to AMP, which results in a production of two molecules of ADP (first reaction). The two molecules of ADP produced in this first reaction receive a phosphate group from polyphosphate by the action of PPK, resulting in a production of two molecules of ATP (second reaction). The two molecules of ATP produced in this second reaction are used again for the first reaction to produce four molecules of ADP, and these four molecules of ADP are then converted into four molecules of ATP by PPK.
In this manner, according to Japanese Laid-Open Patent Publication No. 2001-299390, excess amounts of AMP and polyphosphate are added to the reaction system to drive the ADK and PPK equilibrium toward the production of ADP (first reaction) and the production of ATP (second reaction), respectively. Then, by repeating a single reaction system including the first reaction and the second reaction n times, one molecule of ATP is amplified to 2n molecules of ATP. Therefore, this method is an excellent method for amplifying ATP.
Although this method described in Japanese Laid-Open Patent Publication No. 2001-299390 is an excellent method in that the presence of cells can be detected with a higher level of sensitivity than conventionally achieved, it turned out that in this method, amplification of ATP in the absence of ATP, which does not occur in theory, is sometimes observed at a low level, and thus there is a problem in that this method does not have reliability for amplifying and detecting exogenous (externally added) ATP only. That is to say, there is a problem in that this method cannot reliably provide such a sensitivity that ATP at a single cell level can be amplified and detected. Furthermore, there are also problems such as the adjustment between the activities of ADK and PPK.